Nucleotide sequencing via repetitive single molecule hybridization

ABSTRACT

Methods of obtaining sequence information about target oligonucleotides by repetitive single molecule hybridization are disclosed. The methods include exposing a target oligonucleotide to one or more copies of a test oligonucleotide; measuring hybridization; dehybridizing the test oligonucleotide; and repeating until the information content from the hybridization trials equals or exceeds the information content of the target oligonucleotide.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/896,897 filed on Sep. 6, 2007, which is a continuation of U.S. patentapplication Ser. No. 10/990,939 filed on Nov. 17, 2004, which claims thebenefit of U.S. Provisional Patent Application No. 60/520,751 filed onNov. 17, 2003. The contents of the above applications are incorporatedherein by reference.

BACKGROUND

The advent of the first reference sequence of the human genome by Lander(Nature (15 Feb. 2001) 409: 860-921) and Venter (Science (16 Feb. 2001)291:1304) has generated increased interest in the ability to sequenceentire genomes which range in size from ^(˜)1 megabase to as high as 600gigabases in some organisms. To make uses of the human genome referencesequence tractable, several important innovations including highlyparallel capillary electrophoresis were developed in order to bring basesequencing costs down.

Unfortunately, to go beyond a small number of reference sequences to thepoint where it is feasible to sequence each individual genome in apopulation or to sequence ab initio a large class of new organisms,vastly faster and less expensive means are required. Towards that endseveral new approaches have been proposed and demonstrated to variousdegrees including: Edman degradation and fluorescent dye labeling of asingle DNA strand in a flow cytometer; “sequencing by hybridization”(Perlegen Corp.; Callida Genomics) and “sequencing by synthesis” (Quakeet al., Cal. Tech.; Solexa Corp.). The latter two approaches afford ahigh degree of parallel information retrieval, leveraging chip-basedimaging system approaches to simultaneously record data from a verylarge number of gene chip pixels. Unfortunately, each approach suffersseveral shortcomings.

“Sequencing by hybridization” requires an inordinately large array ofexplicitly patterned spots to approximate the information content of thegenome to be sequenced. In addition, oligonucleotides from the sampleare required to search a very large array of gene chip oligonucleotidecomplements, making hybridization time very long.

“Sequencing by synthesis” avoids a number of the issues above butintroduces difficult and error-prone chemistries which may be difficultto scale effectively.

SUMMARY OF THE INVENTION

The present invention provides “repetitive single moleculehybridization,” which has the ability to carry out de novo sequencing ofDNA, such as genomic DNA, at high speed and low cost.

The invention involves obtaining sequence information from a targetnucleotide. A plurality of target nucleotides can be interrogated inparallel using the methods described herein, permitting the sequencingof an entire genome or a subset thereof. Generally, testoligonucleotides from a library of test oligonucleotides of uniformlength are exposed to one or more target oligonucleotides underconditions permitting hybridization of the test oligonucleotides to aperfectly complementary sequence. The target nucleotide can beimmobilized on a chip such as an inverse gene chip representing a genomeor a portion thereof. A suitable inverse gene chip can be prepared, forexample, by dehybridizing (denaturing) nucleic acids (e.g. by increasingthe temperature or altering solvent conditions), selecting a singlestrand, cutting the strand into target oligonucleotides of averagelength N nucleotides and chemically attaching the target nucleotides toa substrate.

To perform sequencing, a target oligonucleotide is exposed underhybridizing conditions to a first set of identical test oligonucleotidesdrawn from a library of all possible test oligonucleotides of length M.Hybridization or failure to hybridize at the single molecule level isdetected and recorded. The molecules are denatured to separate anyannealed oligonucleotides and the first set of test oligonucleotides isthen washed away. The process is repeated, substituting different setsof identical test oligonucleotides, until the library of testoligonucleotides has been exhausted or until the information content ofthe set of hybridization experiments equals or exceeds the targetedsequence information.

The present invention permits highly parallel sequencing of targetnucleic acids while requiring a library of test oligonucleotides of onlymodest size. Accordingly, in one embodiment, where the targetoligonucleotides are less than 50 nucleotides in length (i.e. N<50),test oligonucleotides having 2, 3, or 4 potentially informativepositions (i.e. the oligonucleotide length, subtracting uninformativespacer positions, universal bases, positions hybridizing to any primersequences incorporated in the target oligonucleotides, or the like) areused; where the target oligonucleotides are 50-99 nucleotides in length,test oligonucleotides having 3, 4, or 5 potentially informativepositions are used; where the target oligonucleotides are 100-999nucleotides in length, test oligonucleotides having 4-6 potentiallyinformative positions are used; where the target oligonucleotides are1,000-10,000 nucleotides in length, test oligonucleotides having 5-8potentially informative positions are used; and where the targetoligonucleotides are greater than 10,000 nucleotides in length, testoligonucleotides having 7-13 potentially informative positions are used.In another embodiment, positive hybridization outcomes are used todeduce a set of possible target oligonucleotide sequences and negativehybridization outcomes are used to eliminate members from the set.

To address the existence of repeats in some nucleic acids of interest,the invention also provides methods of sequencing using testoligonucleotides incorporating one or more spacers separating at leasttwo of the potentially informative positions of the testoligonucleotides. Useful spacers include, for example, double-strandedportions of the test oligonucleotide; spacers comprising an abasicfuran; or other traditional chemical spacers such as those incorporatinga polyethylene glycol portion or an extended carbon chain including, forexample, at least three methylene groups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of preparation of an inverse genechip.

FIG. 1B is a close-up schematic of an inverse gene chip consisting of Koligonucleotides of average length N derived from target genome disposedat random fixed locations.

FIGS. 2A-2E schematically illustrate the steps involved in deriving denovo sequence information from an inverse gene chip by means of repeatedtest hybridizations with members of the library of all possibleoligonucleotides of length M (“M-mers”). FIG. 2A depicts the initialcondition before incubation with test M-mers. FIG. 2B depictsintroduction of a test M-Mer oligonucleotide with fluorescent dye underselective hybridizing conditions and depicts the subsequent parallelreadout with an electronic imager. FIG. 2C depicts the status followingdehybridization and wash steps. FIG. 2D depicts the introduction asecond test M-Mer oligonucleotide under selective hybridizing conditionsand depicts the subsequent parallel readout with the electronic imager.FIG. 2E is a schematic depiction of an inverse DNA chip with multiplecopies of target N-mer oligonucleotides.

FIG. 3 is a schematic example showing the extraction of 16 bits ofhybridization information sufficient for uniquely determining the 6 bitsof sequence information of the target 3-mer.

FIG. 4 is a schematic of an M-mer oligonucleotide interposed with anR-mer double stranded spacer.

FIG. 5 is a table of M-mer library size as a function of M.

FIG. 6A is a graphical representation of information content in bits perhybridization trial as a function of M where N is fixed at 100.

FIG. 6B is a table of optimal values of M for various values of targetoligonucleotide length N.

FIG. 6C is a table of maximum and optimal target oligonucleotide sizes Nfor various values of test oligonucleotide length M.

FIG. 7 schematically depicts an example of a procedure for recoveringsequence information.

FIG. 8 is a flowchart depicting an algorithm for recovering sequenceinformation.

DETAILED DESCRIPTION OF THE INVENTION

An inverse gene chip is known in the field and has been employed in thetechnique termed “sequencing by synthesis” (see Quake et al., PNAS100:3960; Solexa Corp.). FIG. 1A illustrates an exemplary technique forproducing a gene chip. A cell 10 is lysed and its genomic DNA separated.In many cases (other than for single-stranded DNA viruses) the DNA willbe in double-stranded form. The DNA may be dehybridized either usingheat (melting) or chemically or enzymatically to isolate a single strandof the genomic DNA. The genomic DNA is then cut into oligonucleotides ofaverage size N. In addition to other techniques known in the literature,one can create a chimera consisting of an oligonucleotide coupled to anuclease. The chimera will bind periodically to the DNA and cut locally,thus processing the single stranded genomic DNA into a series ofoligonucleotide fragments 20. We refer to the genomic oligonucleotidefragments 20 as target fragments. These target fragments are thenimmobilized on a support by standard 3′ or 5′ coupling chemistry to forman inverse gene chip 30. FIG. 1B is a schematic close-up view of aninverse gene chip 30 consisting of K target oligonucleotides 20 ofaverage length N nucleotides. K is varied based on the amount of nucleicacid sequence information desired and the average length N of thenucleotides; for maximum throughput, however, K is selected to approachthe resolving capacity of the imaging system used.

FIGS. 2A-2E schematically illustrate the steps involved in obtaining thedata required to extract de novo sequence information from DNA. FIG. 2Adepicts the initial condition before incubation with test M-Mers. FIG.2B depicts introduction of a test M-Mer oligonucleotide 25 withfluorescent dye under selective hybridizing conditions and subsequentparallel readout with a conventional electronic imager 40. Electronicimager 40 can incorporate a lens 45 to image inverse gene chip 30 or canoperate without a lens, in which case electronic imager 40 is located inclose proximity to inverse gene chip 30. FIG. 2C shows the conditionsafter a subsequent dehybridization and wash step; the oligonucleotidefragments return to the unbound state as previously shown in FIG. 2A.FIG. 2D depicts the introduction of a second test M-Mer oligonucleotide25 under selective hybridizing conditions and subsequent parallelreadout with electronic imager 40. Readout involves detecting thepresence or absence of a fluorescent signal for each position of inversegene chip 30 associated with a target oligonucleotide 20 and recordingthe presence or absence of a fluorescent signal, generally in acomputer-readable medium. Steps c and d are repeated with subsequentM-mer oligonucleotides until sufficient information is extracted.

Referring to FIG. 2E, it may be convenient in certain cases not torequire single molecule hybridization detection. In this case aplurality of co-located (i.e. located in physical proximity to eachother) copies of target oligonucleotides 20 can be used. Such multipleco-located target oligonucleotides 20 can be readily generated by, forexample, local PCR such as that enabled by rolling circlepolymerization.

FIG. 3 is a schematic example showing the extraction of 16 bits ofhybridization information sufficient for uniquely determining the 6 bitsof sequence information of the target 3-mer 20. There are 42 (sixteen)possible 2-mer test oligonucleotides 25, each of which is sequentiallyincubated with the target 3-mer 20, in this case the nucleotide sequenceC-G-A. Only in cases 8 and 15 is hybridization successful; the positiveand negative hybridization outcomes of all sixteen exposure events aredetected and recorded. If, for example, one were to write a bit sequenceconsisting of 0's in the case of no hybridization and 1's in the case ofsuccessful hybridization for each of the sixteen possible testoligonucleotides 25, then one would have the bit sequence0000000100000010. This bit sequence, representing all possiblehybridization trials from the 2-mer library contains approximately 16bits of information (I explain below why this is only an approximateinformation measure), which is larger than the information content ofthe target 3-mer sequence, CGA, which contains log₂(4³)=6 bits ofinformation. Thus, in most cases a 2-mer library can uniquely decode a3-mer target sequence. In contrast, a palindromic sequence such as CGCcannot be uniquely decoded using only a 2-mer library because both itand the sequence GCG bind the same set of 2-mers (namely CG and GC).Such cases can be disambiguated by applying M-mer libraries of differentsize coupled with the ability to determine the number of testoligonucleotides bound to a given target oligonucleotide 20. Forinstance, in this case, applying a 1-mer library disambiguates CGC fromGCG, as 2 G's would bind to the former and only one would bind to thelatter.

The procedure for determining sequence information about targetoligonucleotide 20 from the test bit sequence requires ordering in anoverlapping manner the test oligonucleotides that successfullyhybridized. Thus we know from the test data that the sequence of targetoligonucleotide 20 has both a CG and a GA with the only overlap at G,giving the unique sequence of CGA. The section discussing FIGS. 7 and 8details the procedure and flowchart algorithm for determining sequencesfrom test hybridization data.

Referring to FIG. 4, there are areas of certain genomes with long repeatsequences, longer than the sizes of target oligonucleotides 20 that canbe conveniently handled by a typical M-mer library. In such cases the Minterrogating nucleotides of test oligonucleotide 25 may be interspacedwith a spacer of length R as depicted in FIG. 4. This allows M-mer 20 tobridge larger target oligonucleotides 20. By combining data obtainedwith M-mer libraries containing different spacer values R full sequencedata can be obtained. The spacer may consist of double strandednucleotide sequences as shown in FIG. 4 which do not interact withtarget oligonucleotide 20, or it may contain a minimally interactingsingle stranded sequence, such as a sequence comprising one or moreabasic furans, or a sequence to which is bound another hybridizationblocker such as a sequence-specific protein binder or asequence-specific zinc finger complex. Exemplary spacers can, forexample, be constructed by incorporating one of the following moietiesduring synthesis of a test oligonucleotide 25:3,6,9-trioxaundecane-1,11-diisocyanate;5′-O-dimethoxytrityl-1′,2′-dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;3-(4,4′-dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;9-O-dimethoxytrityl-triethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;12-(4,4′-dimethyoxytrityloxy)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;or 18-O-dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. Alternatively, thetarget oligonucleotides themselves can have an R-mer cut out of them,where R corresponds to a length of the target oligonucleotide known tohave repetitive or uninformative nucleotide positions.

FIG. 5 is a table of the size of the M-mer library of testoligonucleotides as a function of M assuming the nucleotide at anyposition is chosen from a possible set of 4. The size of the library issimply the number of all possible oligonucleotides of length M which forthe case of h different types of nucleotides is given by h^(M). For mostnatural systems h=4 (i.e. adenine, guanine, cytosine, and eitherthyrnine or uracil). For many applications it will be useful to delivertest oligonucleotides to the inverse gene chip array by means ofmicrofluidics. Practical considerations of delivering testoligonucleotides, time to deliver them as well as time and cost togenerate the library coupled with the number of elements in a singlemolecule hybridization detection system make library sizes of M 8 a goodchoice.

FIGS. 6A-6C relate to the information content of hybridizationexperiments of an M-mer test library on an N-mer target oligonucleotide.Knowledge of the information content is a guide to appropriate andpreferred values for M in order to obtain full de novo sequenceinformation from an N-mer.

The probability of fully hybridizing a random M-mer somewhere on a givenN-mer is given by

P(M,N)=1−[1−(1/h)^(M)]^((N−M+)1)  Eq. 1

where for the case of 4 nucleotides we have h=4.

The information content in bits extractable per test hybridization isgiven by

as derived from analysis used to analyze the statistics of theinformation content from a series of weighted coin flips as is known inthe art. Thus the total number of bits extractable from the h^(M)possible hybridization trials is given by

I(M,N)h^(M)  Eq. 3

The information content in bits of an N-mer oligonucleotide is given by

Log₂(h^(N)  Eq. 4)

In order to obtain full de novo sequence information of a given targetN-mer oligonucleotide we thus require that:

I(M,N)×t Log₂(h^(N))  Eq. 5

where t is the number of hybridization trials noting that a maximum oft=h^(M) independent trials are possible.

FIG. 6A is a plot of the information content in bits per hybridizationtrial as a function of M. As shown there is an optimal M (labeled M*)which yields the highest information content per trial. Too small an Mor too large an M yields non-optimal information content per trial.

FIG. 6B gives M* for various values of the target oligonucleotide lengthN. Since one is limited to h^(M) hybridization trials, an ideal strategyis to employ all possible M* trial hybridizations and then to employ asufficient number of M*+1 trials (i.e. trials using test oligomers oflength M*+1) such that the information content of the hybridizationtrials equals or exceeds the information content of the targetoligonucleotide which one desires to sequence. Accordingly, when theaverage or maximum length of the target oligonucleotides 20 to besequenced is less than 50, M is advantageously 2-4; when N is 50-99, Mis advantageously 3-5; when N is 100-999, M is advantageously 4-6; whenN is 1,000-10,000, M is advantageously 5-8; and when N is greater than10,000, M is advantageously 7-13.

FIG. 6C is a table calculating N_(max), defined as the largest N suchthat I(M,N)4^(M)>2N for various values M of the size of the testoligonucleotides. N_(max) represents the largest possible N that can besequenced by means of the data set derived from sequentially testing thehybridization of all possible M-mers on the target N-mer. Althoughtarget oligonucleotides are most often no more than 10,000 nucleotidesin length (e.g. tens, hundreds, or thousands of nucleotides as shown inFIG. 6C), their length is in fact limited only by the constraints of Eq.5.

An additional parameter, N_(Optimal), which is defined as MAXIMUM[I(M,N)4^(M)−2N], is also calculated. N_(Optimal) is the value of N whichmaximizes the difference in information content between thehybridization trials and the target oligonucleotide of size N.

Referring to FIGS. 7 and 8 an example procedure and flowchart algorithmfor inverting the series of hybridization trials and deriving de novosequence information are given.

Referring to FIG. 7, the example given is the de novo sequencedetermination of a target 7-mer 20 using 3-mer test oligonucleotides 25.According to Eq. 4 the information content of an 7-mer is 14 bits.According to Eq. 2 the information content per hybridization trial of a3-mer on a 7-mer is 0.4 bits and thus according to Eq. 5 on average atleast 40 test oligonucleotides 25 would need to be trial hybridized totarget oligonucleotide 20 to derive the de novo sequence information oftarget oligonucleotide 20.

FIG. 7 gives an example for the general procedure for inverting thesequence showing how both positive hybridization outcomes and negativehybridization outcomes are used to determine sequence. In step 1 anumber of test hybridizations as discussed above are carried out.Whether the test oligonucleotide 25 hybridizes or does not is recorded.In step 2 the test oligonucleotide sequences which are recorded to havesuccessfully hybridized are themselves (or as shown in the figure theircomplement) arrayed into possible target oligonucleotides. This arrayingoperation is generally carried out in silico although it is possible tocarry it out using the techniques of DNA computing known in theliterature. As shown in FIG. 7 possible target oligonucleotides whichwere created with the largest number of overlaps (in this case the mostinstances of two-base overlaps as opposed to single-base overlaps) arethe most likely. In the case of this example there are two possibletarget oligonucleotides ACGGCCA and CCACGGC which both have beenassembled from the set of positively hybridizing test oligonucleotides25 with two instances of double base overlap and one instance of singlebase overlap. The two possibilities thus cannot be disambiguated bymeans of positive hybridizing data alone. In the final step negativehybridizing data, namely data for the set of test oligonucleotides 25which did not successfully bind to the target oligonucleotide, are usedto eliminate possible target oligonucleotide sequences and thus toisolate the correct target oligonucleotide sequence. In this exampledata that show that test oligonucleotide GTG does not bind targetoligonucleotide 20 is used to exclude CCACGGC as a possible targetoligonucleotide (otherwise GTG would be expected to hybridize to the CACportion of CCACGGC) and thus identifies ACGGCCA uniquely as desiredtarget oligonucleotide 20.

FIG. 8 shows a representative series of steps for deriving de novosequence information from a target oligonucleotide using the set of data(both positive and negative) from a sequence of test oligonucleotidehybridization trials. Those steps include:

selecting an M-Mer test oligonucleotide library as described above;

carrying out sufficient sequential hybridization trials (each includingtest hybridization of an M-mer test oligonucleotide to an N-mer targetoligonucleotide, measuring the positive or negative outcome of thehybridization trial, and, before any subsequence test hybridization,dehybridizing and washing) of members of the M-mer test oligonucleotidelibrary with the N-mer target oligonucleotide such that thehybridization trial information content equals or exceeds theinformation content of the desired target oligonucleotide;

using positive hybridization outcomes to construct the possible set oftarget oligonucleotide sequences weighted by the highest degree ofoverlap; and

using negative hybridization outcomes to eliminate members of thepossible set of target oligonucleotide sequences.

This set of steps determines the sequence of each individual targetoligonucleotide on an inverse DNA chip. In order to get the sequence ofthe source nucleotide polymer from which the target oligonucleotideswere derived (e.g. of an entire genome) a second inverse gene chip fromthe same genome can be created in which the target oligonucleotides arecut in a different place, preferably far from the original cuts. Targetoligonucleotide sequences from both inverse DNA chips may now beassembled in a manner similar to that shown in FIG. 7. Again wholegenome sequences created from target oligonucleotide assemblies withmaximum overlap are given highest ranking.

Example 1 Human Genome

As an example of repetitive single molecule hybridization, we considerthe human genome, which includes about 3 billion nucleotide base pairs.Referring to FIG. 6C we choose the 5-Mer test oligonucleotide librarywith an optimal N-mer size of: ^(˜)200 bases. This leads to 15×10⁶ spots(i.e. K=15×10⁶) equivalent to the number of imaging elements availablein current solid state imaging devices or a small array of such imagingdevices. Assuming cycle times (hybridization+measurement+wash) of 5minutes per test oligonucleotide yields a total run time of (1024 testoligonucleotides in the 5-mer test oligonucleotide library)*(5 minutesper cycle)=3.5 days.

Example 2 De Novo Synthesis of Nucleic Acids

Regarding another application of the sequencing technology describedherein, there has recently emerged considerable interest in thesynthesis of long sequences of nucleic acids (See Smith, Hutchison,Pfannkoch, and Venter et al., “Generating a Synthetic Genome by WholeGenome Assembly: phiX174 Bacteriophage from Synthetic Oligonucleotides”PNAS 2003). One approach is the chemical synthesis of oligonucleotidesfollowed by their ligation. Unfortunately the error rate for such anapproach is equivalent to the chemical synthetic error rate of^(˜)1:10². One approach is to sequence the ligated product and thenperform site directed mutagenesis to correct errors. But as describedabove this approach is hampered by throughput and cost of currentelectrophoresis-based sequencing methodologies.

The present invention facilitates accurate synthesis of nucleic acidsaccording to steps comprising:

synthesizing a nucleotide sequence de novo;

a) obtaining sequence information about the synthesized nucleotidesequence by repetitive single molecule hybridization as described above;and

c) using site directed mutagenesis to correct at least one error in thesynthesized nucleotide sequence.

The terms and expressions employed herein are used as terms ofdescription and not of limitation, and there is no intention, in the useof such terms and expressions, of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed.

1. A method of synthesizing de novo a nucleic acid sequence, the methodcomprising the steps of: a) synthesizing a nucleic acid sequence denovo; b) obtaining information about the nucleic acid sequence byrepetitive single molecule hybridization, thereby to detect an error inthe nucleic acid sequence; and c) correcting the error in the nucleicacid sequence by site-directed mutagenesis, thereby synthesizing de novothe nucleic acid sequence.
 2. The method of claim 1, wherein step b) iseffected by: (a) hybridizing one or more copies of a testoligonucleotide comprising at least two informative region sequences toa single molecule of target oligonucleotides under conditions permittinghybridization of the test oligonucleotide to a perfectly complementarysequence; (b) measuring a signal of said hybridization of the testoligonucleotide to the target oligonucleotide, thereby completing afirst hybridization trial; (c) dehybridizing the test oligonucleotide;(d) exposing the target oligonucleotide to one or more copies of atleast one additional test oligonucleotide, said additional testoligonucleotide comprising at least two informative regions; (e)measuring a signal from the hybridization of the additional testoligonucleotide to the target oligonucleotide, thereby completing afurther hybridization trial; (f) repeating steps c) to e) until theinformation content from the hybridization trials exceeds theinformation content of the target oligonucleotide; and (g) analyzingsaid signals.
 3. The method of claim 2, wherein said signal of saidhybridization comprises a fluorescent tag.
 4. The method of claim 2,wherein said signal of said hybridization comprises a quantum dot ornanoparticle tag.
 5. The method of claim 2, wherein said measuring iseffected mechanically.
 6. The method of claim 2, wherein said measuringis effected by sensing a change in charge associated with hybridization.7. The method of claim 2, wherein said measuring is effectedelectronically.
 8. The method of claim 2, wherein said targetoligonucleotide is immobilized on a solid support.
 9. The method ofclaim 2, wherein a plurality of said target oligonucleotides areimmobilized on a solid support.
 10. The method of claim 1, wherein step(b) is effected by: (a) hybridizing one or more copies of a testoligonucleotide to a single molecule of target oligonucleotides underconditions permitting hybridization of the test oligonucleotide to aperfectly complementary sequence; (b) measuring a signal of saidhybridization of the test oligonucleotide to the target oligonucleotide,thereby completing a first hybridization trial; (c) dehybridizing thetest oligonucleotide; (d) exposing the target oligonucleotide to one ormore copies of at least one additional test oligonucleotide; (e)measuring a signal from the hybridization of the additional testoligonucleotide to the target oligonucleotide, thereby completing afurther hybridization trial; (f) repeating steps c) to e) until theinformation content from the hybridization trials exceeds theinformation content of the target oligonucleotide; and (g) analyzingsaid signals; and (h) analyzing an absence of said signals to eliminateoligonucleotide test members.
 11. The method of claim 10, wherein saidsignal of said hybridization comprises a fluorescent tag.
 12. The methodof claim 10, wherein said signal of said hybridization comprises aquantum dot or nanoparticle tag.
 13. The method of claim 10, whereinsaid measuring is effected mechanically.
 14. The method of claim 10,wherein said measuring is effected by sensing a change in chargeassociated with hybridization.
 15. The method of claim 10, wherein saidmeasuring is effected electronically.
 16. The method of claim 10,wherein said target oligonucleotide is immobilized on a solid support.17. The method of claim 10, wherein a plurality of said targetoligonucleotides are immobilized on a solid support.